1. Technical Field
The field of this invention is the production of xenogeneic specific binding proteins in a viable mammalian host.
2. Background
The ability to produce transgenic animals has been revolutionized with the advent of the ability to culture murine embryonic stem cells, and to introduce genetic modifications in these cells for subsequent transmission to the mouse germline. Thus one has the opportunity to modify endogenous genes to produce animal strains capable of producing novel products by introduction of foreign genes into the host, particularly human genes to produce xenogeneic binding proteins. The expression of such genes in vivo in an animal model may provide for investigation of the function of the gene, the regulation of gene expression, its processing, response to various agents and the like. In addition, animals with new phenotypes, including those that mimic a variety of diseases, may be produced. For example, there is interest in introducing a dominant mutation or complementing a recessive mutation. Depending on the particular gene, the difficulty of achieving the desired mutation will vary greatly. While some gene targets have proven to be relatively amenable to modification, other targets have proven to be extremely resistant to modification.
Because of the opportunity for generating transgenic animals, there is substantial interest in providing new procedures that increase the success of production of transgenic animals. Particularly, where one wishes to introduce large DNA fragments, encompassing hundreds of kilobases, there is substantial concern about the ability to introduce the large fragments in intact form into mammalian cells, the efficiency of integration, the functional capability of the gene(s) present on the fragment and transmission in the germline to the progeny. In addition, such procedures for introduction of large DNA fragments provide for determination of the function of large DNA fragments identified in the ongoing human genome project.
In particular, there is interest in producing xenogeneic specific binding proteins, for example human monoclonal antibodies, in small laboratory animals such as mice. Monoclonal antibodies find use in both diagnosis and therapy. Because of their ability to bind to a specific epitope, they can be uniquely used to identify molecules carrying that epitope or may be directed, by themselves or in conjunction with another moiety, to a specific site for diagnosis or therapy.
Monoclonal antibodies comprise heavy and light chains which join together to define a binding region for the epitope. Each of the chains is comprised of a variable region and a constant region. The constant region amino acid sequence is specific for a particular isotype of the antibody, as well as the host which produces the antibody.
Because of the relationship between the sequence of the constant region and the species from which the antibody is produced, the introduction of a xenogeneic antibody into the vascular system of the host can produce an immune response. Where the xenogeneic antibody is introduced repetitively, in the case of chronic diseases, it becomes impractical to administer the antibody, since it will be rapidly destroyed and may have an adverse effect. There have been, therefore, many efforts to provide a source of syngeneic or allogeneic antibodies. One technique has involved the use of recombinant DNA technology where the genes for the heavy and light chains from a host were identified and the regions encoding the constant region isolated. These regions were then joined to the variable region encoding portion of other immunoglobulin genes from another species directed to a specific epitope.
While the resulting chimeric partly xenogeneic antibody is substantially more useful than using a fully xenogeneic antibody, it still has a number of disadvantages. The identification, isolation and joining of the variable and constant regions requires substantial work. In addition, the joining of a constant region from one species to a variable region from another species may change the specificity and affinity of the variable regions, so as to lose the desired properties of the variable region. Also, there are framework and hypervariable sequences specific for a species in the variable region. These framework and hypervariable sequences may result in undesirable antigenic responses.
It would therefore be more desirable to produce allogeneic antibodies for administration to a host by immunizing the host with an immunogen of interest. For primates, particularly humans, this approach is not practical. The human antibodies which have been produced have been based on the adventitious presence of an available spleen, from a host which had been previously immunized to the epitope of interest. While human peripheral blood lymphocytes may be employed for the production of monoclonal antibodies, these have not been particularly successful in fusions and have usually led only to IgM. Moreover, it is particularly difficult to generate a human antibody response against a human protein, a desired target in many therapeutic and diagnostic applications. There is, therefore, substantial interest in finding alternative routes to the production of allogeneic antibodies for humans.
Thomas and Capecchi (1987), Cell, 51:503-512 and Koller and Smithies (1989), Proc. Natl. Acad. Sci. USA, 86:8932-8935 describe inactivating the xcex22-microglobulin locus by homologous recombination in embryonic stem cells. Berman et al. (1988), EMBO J. 7:727-738 describe the human Ig VH locus. Burke, et al. (1987), Science, 236:806-812 describe yeast artificial chromosome vectors. See also, Garza et al. (1989), Science, 246:641-646 and Brownstein et al. (1989), Science, 244:1348-1351. Sakano, et al., describe a diversity segment of the immunoglobulin heavy chain genes in Sakano et al. (1981), Nature, 290:562-565. Tucker et al. (1981), Proc. Natl. Acad. Sci. USA, 78:7684-7688 describe the mouse IgA heavy chain gene sequence. Blankenstein and Kruwinkel (1987), Eur. J. Immunol., 17:1351-1357 describe the mouse variable heavy chain region. See also, Joyner et al. (1989), Nature, 338:153-155, Traver et al. (1989), Proc. Nat. Acad. Sci. USA 86:5898-5902, Pachnis et al. (1990), Proc. Nat. Acad. Sci. USA, 87:5109-5113 and PCT application PCT/US91/00245. Bruggemann et al., Proc. Nat. Acad. Sci. USA; 86:6709-6713 (1989); Behring Inst. Mitt. 87:21-24 (1990); Eur. J. Immunol. 21:1323-1326 (1991), describe monoclonal antibodies with human heavy chains. Albertsen et al., Proc. Nat. Acad. Sci. USA 87:4256-4260 (1990), describe the construction of a library of yeast artificial chromosomes containing human DNA fragments. Yeast artificial chromosome vectors are described by Burke et al., Science 236:806-812 (1987). Pavan et al., Mol. and Cell. Biol. 10(8):4163-4169 (1990) describe the introduction of a neomycin resistance cassette into the human-derived insert of a yeast artificial chromosomes using homologous recombination and transfer into an embryonal carcinoma cell line using polyethylene glycol-mediated spheroplast fusion. Pachnis et al., Proc. Nat. Acad. Sci. USA 87:5109-5113 (1990), and Gnirke et al., EMBO Journal 10(7):1629-1634 (1991), describe the transfer of a yeast artificial chromosome carrying human DNA into mammalian cells. Eliceiri et al., Proc. Nat. Acad. USA 88:2179-2183 (1991), describe the expression in mouse cells of yeast artificial chromosomes containing human genes. Huxley et al., Genomics 9:742-750 (1991) describe the expression in mouse cells of yeast artificial chromosomes containing the human HPRT.gene. Mortensen et al., Mol. and Cell. Biol. 12(5):2391-2395 (1992) describe the use of high concentrations of G418 to grow heterozygous embryonic stem cells for selection of homozygous mutationally altered cells. Yeast protoplast fusion with mouse fibroblasts is described by Traver et al., Proc. Nat. Acad. Sci. USA 86:5898-5902 (1989) and Pachnis et al., Proc. Nat. Acad. Sci. USA 87:5109-5113 (1990). Davies et al., Nucl. Acids Res. 20:2693-2698 (1992) describe targeted alterations in YACs. Zachau, Biol. Chem. 371:1-6 (1990) describes the human immunoglobulin light (kappa) (IgK) locus; Matsuda et al., Nature Genetics 3:88-94 (1993) and Shin et al., EMBO 10:3641-3645 (1991) describe the cloning of the human immunoglobulin heavy (IgH) locus in YACs.
Xenogeneic specific binding proteins are produced in a non-human viable host by immunization of the host with an appropriate immunogen.
A preferred non-human host is characterized by: (1) being incapable of producing endogenous immunoglobulin heavy chain; (2) being substantially incapable of producing endogenous immunoglobulin light chains; and (3) capable of producing xenogeneic immunoglobulin light and heavy chains to produce a xenogeneic immunoglobulin or immunoglobulin analog. Thus, the host may have an entire endogenous immunoglobulin locus substituted by a portion of, or an entire, xenogeneic immunoglobulin locus, or may have a xenogeneic immunoglobulin locus inserted into a chromosome of the host cell and an inactivated endogenous immunoglobulin region. These various alternatives will be achieved, at least in part, by employing homologous recombination for inactivation or replacement at the immunoglobulin loci for the heavy and light chains.
Additionally, novel methods are provided for introducing large segments of xenogeneic DNA of at least 100 kb, particularly human DNA, into host animals, particularly mice, by introducing a yeast artificial chromosome (YAC) containing a xenogeneic DNA segment of at least 100 kb, into an embryonic stem cell for integration into the genome of the stem cell, selection of stem cells comprising the integrated YAC by means of a marker present in the YAC, introduction of the YAC-containing ES cells into embryos and generation of chimeric mice from the embryos. The chimeric animals may be mated to provide animals that are heterozygous for the YAC. The heterozygous animals may be mated to generate progeny homozygous for the integrated YAC.